We have been studying the role of molecular chaperones in DNA replication and proteolysis. Molecular chaperones are proteins that fold, unfold and refold other proteins without themselves being part of the final protein complex. Chaperones are present in all organisms, are highly conserved and many are induced by environmental stresses such as heat shock. One chaperone that we have been studying is DnaK, the major 70 kDa heat shock protein of E. coli. We discovered that DnaK and two cochaperones, DnaJ and GrpE, activate origin specific DNA binding by the plasmid P1 initiator protein, RepA. We found that activation is the conversion of inactive RepA dimers to active RepA monomers that bind with high affinity to the P1 origin of replication. We used the P1 RepA activation reaction as a model system to look for other molecular chaperones and discovered that ClpA, the ATPase component of the ATP-dependent ClpAP protease, is a molecular chaperone belonging to the ubiquitous family of Clp proteins. ClpA performs two known functions of chaperones in vitro: It activates P1 RepA, like the combination of DnaJ, DnaK and GrpE and it prevents heat inactivation of firefly luciferase and RepA. ClpAP degrades RepA, suggesting that an essential energy-dependent step in protein degradation is the targeting and unfolding of substrates for recognition by a specific protease. We have studied the intermediates and products in the RepA activation reaction to elucidate the mechanism of the chaperone function of ClpA. In a first reaction requiring ATP but not ATP hydrolysis, ClpA self assembles into hexamers and makes a complex with inactive RepA dimers. We discovered that one cycle of binding of RepA to ClpA followed by ATP-dependent release converts inactive RepA to active RepA. The activation reaction carried out by ClpA is reversible, suggesting that the chaperone does not covalently modify RepA but rather refolds or remodels it.